Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method (Wiegand et al., 2008 (link)). Exponentially growing precultures were seeded in clear 96-well flat-bottom plates (Greiner Bio-One) at OD600 = 0.05 in the presence of two-fold serial dilutions of assay compounds in a volume of 200 μl/well. Assay plates were sealed (Breath-Easy membrane, Sigma-Aldrich) and incubated for 7 days at 37°C and 80 rpm prior turbidity determination (OD600, Tecan M200Pro plate reader). MIC curves were plotted using the Graph Pad Prism 5 software and the concentration that inhibits 50 and 90% of growth compared to the drug-free control were defined as MIC50 and MIC90, respectively. To establish the Minimum bactericidal concentration (MBC), the colony forming units (CFUs) per assay well were determined by plating serial dilutions of samples onto agar (Murugasu-Oei and Dick, 2000 (link)). Colonies were counted after 3–4 weeks of incubation at 37°C. The MBC was defined as the lowest concentration of test compound that killed 90% of the initial inoculum.
Clear 96 well flat bottom plates
Manufactured by Greiner
Clear 96-well flat-bottom plates are a basic laboratory equipment item used for a variety of scientific applications. They provide a standardized multi-well format with a clear, flat-bottom design.
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Lab products found in correlation
3 protocols using clear 96 well flat bottom plates
1
Determination of Antimicrobial Potency
2
Minimum Inhibitory Concentration Determination
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Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method (Wiegand et al., 2008 (link)). Exponentially growing precultures were seeded in clear 96-well flat-bottom plates (Greiner Bio-One) at OD600 = 0.05 in the presence of two-fold serial dilutions of assay compounds in a volume of 200 μl/well. Assay plates were sealed (Breath-Easy membrane, Sigma-Aldrich) and incubated for 7 days at 37°C and 80 rpm prior to turbidity determination (OD600, Tecan M200Pro plate reader). Percentage growth was determined compared to untreated control and plotted as a function of drug concentration (Graph Pad Prism 5 software). The concentrations that inhibit 90% of growth were recorded as MIC.
3
Peptide Aggregation Screening Assay
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Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from GE Healthcare (Chicago, IL) while all other media and tissue culture reagents such as minimum essential medium (MEM) and phosphate-buffered saline (PBS) were purchased from Gibco, ThermoFisher (Pittsburgh, PA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo-Fisher unless otherwise indicated. Peptide Aggregation Screen Stock solutions of 1018 and derivatives thereof were prepared at 10 mg/ml in water and subsequently diluted into different excipient solutions (a complete list is shown in Table S1). Samples were mixed in clear 96-well flat-bottom plates (Greiner Bio-One, Kremsm€ unster, Austria) to a final peptide concentration of 1 mg/ml and a sample volume of 100 ml. Water was added into each vehicle in parallel to serve as a negative control. Following overnight incubation at 20 C, the optical density at 600nm (OD 600 ) of each sample was recorded on a PowerWave X 340 microplate reader (Bio-Tek Instruments Inc., Winooski, VT). The peptide was considered to aggregate if a specific condition led to an increase in sample turbidity/OD 600 relative to the negative control.
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